Immunometabolic changes indicative of TI in macrophages expressing BRAFV600E. (A) Western blot determination of phosphorylated AKT (p-AKT). The levels of total AKT in whole-cell lysates were used as the protein-loading control. (B) Analysis of ECAR measurements on primary human monocytes isolated from healthy volunteers in 3 different conditions, nontransduced (UT), transduced with wtBRAF (WT) or BRAFV600E (VE), in basal conditions and after sequential addition of glucose, oligomycin, and 2-DG. (C) Relative ECAR to oxygen consumption rate ratio (right). ***P < .001; ****P < .0001, unpaired Student t test. (D) Summary of metabolomics studies: primary human monocytes isolated from 3 healthy volunteers were cultured in 3 different conditions: nontransduced (UT, isolated primary monocytes), transduced with wtBRAF (WT), and transduced with BRAFV600E (VE). Cells were cultured in RPMI-1640+FBS only (Cold) or RPMI-1640+FBS with the addition of 13C6-glucose labeled glucose or 13C5-glutamine for tracing experiments. (E) Principal component analysis (PCA) of high-throughput metabolomics analysis on cold samples showing separation of experimental conditions along the component 1 axis (76.3%); interindividual variability is described by the component 2 axis (12.8%). (F) Hierarchical clustering analysis of the top 50 significant metabolites (Student t test) is represented as a heat map grouped by pathways that are known to be relevant to TI. (G) Overview of the glycolysis pathway in tracing experiments with U-13C6-glucose. (H) Overview of the TCA pathway in tracing experiments with 13C5-glutamine. (I) Incorporation of carbons in de novo synthesized cholesterol: carbon molecules deriving from either labeled glucose (red) or labeled glutamine (blue) are expressed as a percentage of the total. (J) Hematoxylin and eosin stain of ECD lesion (skin biopsy specimen, original magnification x200). (G-I) *P < .05; **P < .01; ***P < .001; ****P < .0001; ns, nonsignificant. Statistical significance of differences was evaluated with ANOVA. FBS, fetal bovine serum.
Figure 4.

Immunometabolic changes indicative of TI in macrophages expressing BRAFV600E. (A) Western blot determination of phosphorylated AKT (p-AKT). The levels of total AKT in whole-cell lysates were used as the protein-loading control. (B) Analysis of ECAR measurements on primary human monocytes isolated from healthy volunteers in 3 different conditions, nontransduced (UT), transduced with wtBRAF (WT) or BRAFV600E (VE), in basal conditions and after sequential addition of glucose, oligomycin, and 2-DG. (C) Relative ECAR to oxygen consumption rate ratio (right). ***P < .001; ****P < .0001, unpaired Student t test. (D) Summary of metabolomics studies: primary human monocytes isolated from 3 healthy volunteers were cultured in 3 different conditions: nontransduced (UT, isolated primary monocytes), transduced with wtBRAF (WT), and transduced with BRAFV600E (VE). Cells were cultured in RPMI-1640+FBS only (Cold) or RPMI-1640+FBS with the addition of 13C6-glucose labeled glucose or 13C5-glutamine for tracing experiments. (E) Principal component analysis (PCA) of high-throughput metabolomics analysis on cold samples showing separation of experimental conditions along the component 1 axis (76.3%); interindividual variability is described by the component 2 axis (12.8%). (F) Hierarchical clustering analysis of the top 50 significant metabolites (Student t test) is represented as a heat map grouped by pathways that are known to be relevant to TI. (G) Overview of the glycolysis pathway in tracing experiments with U-13C6-glucose. (H) Overview of the TCA pathway in tracing experiments with 13C5-glutamine. (I) Incorporation of carbons in de novo synthesized cholesterol: carbon molecules deriving from either labeled glucose (red) or labeled glutamine (blue) are expressed as a percentage of the total. (J) Hematoxylin and eosin stain of ECD lesion (skin biopsy specimen, original magnification x200). (G-I) *P < .05; **P < .01; ***P < .001; ****P < .0001; ns, nonsignificant. Statistical significance of differences was evaluated with ANOVA. FBS, fetal bovine serum.

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